Saving DNA: the Frozen Ark Project

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Essentials

The Frozen Ark is a conservation project which collects and preserves the tissues, cells and DNA of the world’s most endangered animals.

There are a number of other collections of biological material—dubbed ‘frozen zoos’—across the globe.

Human activities are responsible for the rapid disappearance of many animal species.

While resurrecting already-extinct animals such as the thylacine poses enormous technical challenges, properly stored cells and DNA could, in theory, be used to bring back species should they go extinct in the future.

Half a decade ago, Bryan Clarke, a PhD student at the University of Oxford, was stalking snails in the Polynesian islands. With a huge number of snail species, and unique topography—high mountains and low valleys resulting in different species living in relative isolation—it was a sensible place to be doing so. Studying them, Bryan and his team hoped, could give insights into how new species develop. Today, tens of thousands of these samples, collected over 12 years, are housed in the Natural History Museum of London.

What began as a study of speciation, however, was to become something else: a study of extinction.

In the 1950s, giant African land snails, the edible delicacy Achatina fulica, were deliberately introduced to the islands of Tahiti. Perhaps predictably, having no natural predators, they were able to breed like, well, you know. Pretty soon, the giant snails were munching their way through the islands’ crops.

So, how to get rid of them? You guessed it—some bright spark came up with the idea of introducing yet another species of snail. Euglandina rosea like eating other snails: hence their common name, the cannibal snail (aka the rosy wolf snail). These thugs of the mollusc world would, the thinking went, soon clean up the African snail problem, Dexter Morgan–style. In the grand tradition of cane toads and plague minnows, however, it all went horribly wrong. Euglandina rosea ate snails, yes—just not the right ones. Instead of the giant African snails, they went for easier targets—the many species of smaller, native Partula snails. Less filling, maybe, but so much easier to catch. The introduction of Euglandina rosea resulted in the loss of 100 species of snails over around 15 years.

Bryan brought back some of the remaining Partula snails to England and, thanks to some TLC and a diet of porridge, lettuce and tissue paper, was able to breed five of seven species. DNA was also extracted from the snails so that research could be continued in the lab. The conservation and breeding of Partula snails continues today at the Zoological Society of London and other sites around the world.

It was with the collection and preservation of the genetic material of these snail species, and their rescue from complete extinction, that the idea for a comprehensive program of collecting and preserving the cells and DNA of the world’s endangered species was born—what became known as the Frozen Ark Project.

Noah’s Ark on ice

By the 1990s there were already several institutions around the world, such as museums and labs, collecting animal tissue, cells and DNA. Indeed, it was here in Australia, in 1995, that the world’s first national wildlife gene bank was established: the Animal Gene Storage and Resource Centre of Australia (AGSRCA), now known as the Australian Frozen Zoo. Today, the collection holds hundreds of genetic samples from threatened and endangered species from Australia and beyond—from the northern hairy-nosed wombat to the black rhino.

While the Australian Frozen Zoo is suitably housed in liquid nitrogen in a secure facility at Monash University, Victoria, many other collections weren’t in a form suitable for the long-term preservation of undamaged DNA. There was also little global collaboration between the various institutes.

That’s where the Frozen Ark Project, a non-profit independent charity run out of the University of Nottingham in the United Kingdom, came in. It was established in 1994 with the aims of developing global collaboration between such institutions; establishing best practice for the collection and storing of tissues, cells and DNA; and concentrating efforts on the conservation of threatened species. Today the international Frozen Ark Consortium consists of 22 members across the globe: in the UK, the US, Australia, New Zealand, Germany, India, Korea, South Africa, Norway and Ireland. They include the Natural History Museum in London, the American Museum of Natural History, San Diego Zoo Global (which runs the San Diego Zoo) and the Australian Frozen Zoo.

The animal counterpart to plant collections such as the Millennium Seed Bank at Kew Gardens and the Svalbard Global Seed Vault, the Frozen Ark collects and stores the tissues, cells, gametes (sperm and egg cells) and DNA of endangered animals. It has its own collection of around 700 samples, from the scimitar horned oryx to Varroa-mite free honeybees, which it stores in a lab at Nottingham, and also catalogues the samples held in the collections of its members.

The grand vision is to create a collection and database of the tissues, cells and DNA of the world’s endangered animals—before it’s too late.

Deadlier than a giant asteroid?

To date, Earth has seen five mass extinctions—from the ice age which wiped out sea creatures known as graptolites 444 million years ago to the asteroid that’s though to have killed off the dinosaurs around 66 million years ago. Many scientists think we may be in the middle of a sixth. This time, the threat is much closer to home than a giant asteroid. It’s us. Although it can be hard to work out exact figures when it comes to extinction rates, partly because no one knows exactly how many species are out there, it’s estimated that around 100 to 1,000 species (including both animals and plants) per million are lost each year, mostly due to human-caused habitat destruction and climate change. Before humans evolved, the number was less than one species per million per year.

Extinct: no reasonable doubt that the last individual diedCritically Endangered: facing an extremely high risk of extinction in the wildEndangered: facing a very high risk of extinction in the wildVulnerable: facing a high risk of extinction in the wildNear Threatened: is not presently Critically Endangered, Endangered or Vulnerable, but is close to qualifying for or is likely to qualify for a threatened category in the near future.
Source: IUCN Red List 2016

Take Australia. While the thylacine (aka Tasmanian tiger) has the dubious honour of being the poster child of human-caused extinction, it’s just one of a depressingly significant pool of Australian animals to have become extinct since European settlement—many of them unique to this country. The 2016 International Union for the Conservation of Nature (IUCN) Red List cites 31 Australian species known to be extinct, including many small marsupials, such as the desert bettong and desert rat kangaroo, as well as over half a dozen bird species. One hundred and twenty-two Australian species are ‘threatened’ (Critically Endangered, Endangered or Vulnerable), of which 13 are Critically Endangered.

Extinction is a natural process. Ninety-nine percent of the species that have ever lived on Earth are now extinct. But never before in the history of life has just one species been responsible for the extinction of so many others, or in such a short period of time.Sir David Attenborough, The Frozen Ark Project

Future-proofing endangered species

In a perfect world, frozen zoos around the globe would keep an archive of the genetic record of all endangered species. In the absence of unlimited time and resources, the focus of the Frozen Ark Project is on preserving the tissues, cells and DNA of species categorised by the IUCN as Extinct in the Wild—species that no longer roam free in their natural environment, but still exist in zoos.

Conservation is a major focus of the Frozen Ark. Genetic material, such as stored eggs and sperm, could potentially be used in conservation breeding programs. Because inbreeding can become a problem with limited populations living in zoos, for instance, stored sperm might be introduced to diversify the gene pool.

Keeping a genetic record of animal species is important for research, too. When a species become extinct, it takes with it all the information contained in its cells and DNA. Holding within it a record of all the adaptations made in the species over time, DNA can give us insights into the evolution of a species and the genetic relationships between species.

As well, the project carries out research into the best ways of preparing and storing samples. And, in future, it hopes to train researchers around the world in how to collect and freeze biological materials.

If, like me, you were wondering how DNA is actually collected, it’s surprisingly straightforward. You can do it at home with a banana or some split peas. You can even impress your guests with a strawberry DNA daiquiri (Google it—it’s a thing).

It’s essentially the same process (minus the blender and strawberry deliciousness) in the lab. DNA can be collected with no harm to the animal—from its hair follicles, skin or a blood sample. Once collected, the cells are burst open by adding a lysis solution, then placed in warm water. Detergent in the lysis solution disrupts the cell membrane and the envelope around the nucleus, while an enzyme cuts apart the histones (the proteins around which the strands of DNA are wrapped).

A dash of salt is added, which causes proteins and other cellular debris to clump together. A test tube containing the lot then goes into a microcentrifuge—the spinning causing the clumps to sink to the bottom, so that they’re nicely separated from the DNA. Some isopropyl alcohol is added to isolate the concentrated DNA. Then it’s back into the centrifuge for a last spin to send the DNA to the bottom of the test tube. The liquid is removed, and the DNA is allowed to dry.

So far, so simple. Preserving DNA for future generations, though, is another story. Freezing is an effective way of halting the activity of enzymes or chemicals which might cause damage to collected DNA, but sticking it in your home freezer at the usual minus 18 degrees Celsius isn’t going to cut it—at least not for long. For the short-term (days to weeks), DNA can be stored in a salt solution at 4˚ Celsius. If you want it to last for months, it’ll need to go into a salt solution at –80˚ Celsius. To last years, DNA needs to be stored in ethanol at –80˚.

If you want your DNA samples to last decades, they’ll need to be dried and stored on special filter paper known as FTA (standing for Fast Technology for Analysis of nucleic acids) card. Alternatively, you could freeze it in liquid nitrogen: an effective solution, but expensive—and not entirely worry free. Liquid nitrogen needs to be topped up regularly, and tanks can rupture, meaning the potential to lose everything—which would be disastrous for a repository such as a frozen zoo. For this reason, duplicates need to be stored independently.

Oh—and light, bacteria and fungi can also damage DNA, so best to keep your samples in the dark, and free from bacterial or fungal contamination.

Bringing back the dodo?

American evolutionary molecular biologist Beth Shapiro jokes that, when she says what she does for a living, the first question she gets is: can we clone a mammoth? I experienced pretty much the same when mentioning to family and friends that I was writing this article. So—can we?

The focus of the Frozen Ark is preservation, not resurrection. But, for argument’s sake, you may ask, what if a species does go extinct? What then?

First of all, let’s tackle the chestnut of bringing back already-extinct animals such as the mammoth, dodo or thylacine via cloning. Short answer: you can’t. Sorry. For starters, cloning requires a living cell. The idea is to take a cell from the species you want to clone—say, a mammoth hair cell—and trick it into thinking it isn’t a specialised (somatic) cell, but a primordial, unspecialised (stem) cell, able to become any kind of cell in an organism. You then take an unfertilised egg cell (we could perhaps use egg cells from the closely genetically related elephant), and put the two together, so that the elephant egg cell takes on the genetic information of the mammoth. In other words, the genetic information from the nucleus of the mammoth’s somatic cell is transferred to the elephant’s egg cell—hence the name for this process: somatic cell nuclear transfer.

First hurdle, and it’s a pretty intractable one: we don’t have any living somatic cells from mammoths, dodos or thylacines. True, we have intact preserved specimens, but, thanks to time and bacteria, the cells and DNA have decayed somewhat—just as they do for all of us almost from the moment we die.

A second possibility is that we artificially create, or synthesise, DNA, to implant it into the egg of a closely related species. However, even if we can generate the entire DNA sequence, say, of a mammoth or a thylacine (and both are being done right now), that’s just the first step.
DNA doesn’t just slosh around in an inert container to make an animal. First, it must be packaged just right into chromosomes with proteins modified in all sorts of subtle ways so that it is active at the right time and right place. We have no idea how to do this at present.

Then the chromosomes must be folded up correctly inside a nucleus, inside layers of membranes with little holes to modulate what molecules come and go. And this nucleus must be positioned in a cell with all sorts of membranes and organelles to translate the genetic code, provide energy and make sure the products of 20,000 genes interact the right way. Again, we have no idea how to do this at present.

If we could only get as far as synthesising the DNA and packaging it into chromosomes and folding into a nucleus, it’s possible that we could transplant the nucleus into an egg. For instance, thylacine DNA in an artificial nucleus could be squirted into the egg of the (closely genetically related) Tasmanian devil. The resulting embryo would be more or less thylacine (with Tassie devil mitochondria—the genetic information residing outside the nucleus).

Can we substitute man-made DNA for DNA in a living cell? Substitute thylacine for devil DNA and make a new Tasmanian tiger? Yes, but just a tiny bit. At present it’s possible to insert a tiny piece of thylacine DNA (all that’s left from dried skins and formaldehyde-fixed embryos) and insert it into a mouse egg and see it expressed. The embryo, however, is still resolutely mouse, even if it expresses a thylacine protein.

Could we bring back recently extinct animal species from frozen samples? Image source: IStock / JackF.

Okay, so that’s already-extinct animals. But what about those samples collected and stored by the world’s frozen zoos? If the critically endangered Colombian spider monkey (one of the species whose genetic material has been collected by the Frozen Ark) were to disappear, for instance, could we bring it back? In theory, yes. Sadly, there were no Neanderthals running around with vats of liquid nitrogen in which to preserve mammoth cells for posterity. By contrast, the biological material in the world’s frozen zoos has been carefully preserved—and a properly frozen cell is a living cell. Such biological material could be thawed, stem cells made, and implanted in an egg to be part of an embryo.

A bigger question, perhaps, is whether we should bring back the animals that, let’s face it, we’re responsible for having killed off in the first place—the ethics of what’s been dubbed ‘de-extinction’. Dr Ann Clarke, one of the founding members of the Frozen Ark, is of the view that what’s done with genetic material collected by the world’s frozen zoos is for future generations to decide. But better to have the option, she reasons, than not. If current estimates of the number of threatened species are anything to go by, keeping our options open may be a race against time.